Project Summary Enteroviruses are among the most prevalent human pathogens. Traditionally, these viruses were associated with infection of cells within the alimentary tract, oral-fecal transmission between hosts and the development of rare neurological complications including acute flaccid myelitis (AFM). Yet, numerous members of this virus family including human rhinoviruses and enterovirus D-68 (EV-D68) are respiratory pathogens, replicating within the epithelial cells of the upper and lower respiratory tracts, and transmitted between hosts by aerosols In 2014 it was recognized that EV-D68 infection can also result in AFM. With each subsequent biennial outbreak of EV-D68 infection, the number of children infected with EV-D68 has been rising as well as the number of confirmed cases of AFM. Despite strong epidemiologically evidence that the virus is an etiologic agent of AFM, this association has been questioned due the inability to isolate infectious virus from the cerebrospinal fluid of children with EV-D68 induced paralysis. Together, these observations attest to the socioeconomic impact of EV-D68 infection as well as the unmet medical need for better an understanding of EV-D68 biology and the development of novel therapeutics. Notwithstanding the development of multiple animal models of EV-D68 infection, no system replicates the spectrum of pathologies associated with EV-D68; consequently, these models are insufficient for the development and testing of vaccines and antiviral therapies to treat or prevent infection or the development of AFM. We hypothesize that the limited genetic diversity of inbred mice used in many in vivo models of infectious disease limits the establishment of an animal model of EV-D68 biology. The enhanced genetic diversity of mice of the collaborative cross (CC) emulates that of the human population as well as the subtleties in associated phenotypes that are missing in null mice. The goal of the proposed research is to establish a mouse model of EV-D68 pathology and identify naturally-occurring genetic polymorphisms in loci whose products quantitatively regulate susceptibility to viral disease. To identify CC mice susceptible to EV-D68 infection, air-liquid interface cultures of the bronchial epithelium and organotypic brain slice cultures will be generated from 8- and 28-day old mice of randomized genetic backgrounds and infected with three isolates of EV-D68, and the production of infectious virus will be assessed by plaque assay. We will also identify the cell types that are infected by, and visualize tissue damage resulting from, virus infection by H&E staining and indirect immunofluorescence using antibodies generated against the viral protein VP1, and proteins specific to respiratory and neuronal cell types. The mice identified in Aim 1 in which EV-D68 replicated with the highest and lowest efficiency will be crossed in Aim 2. Analysis of the F1 generation will determine the mode by which EV-D68 susceptibility is inherited, while that of the F2 generation will describe the quantitative trait loci (QTL) that modulate EV-D68 infection. Use of CC mice will promote the development of an animal model that reflects the spectrum of pathologies associated with EV-D68 infection.